A recombinant polypeptide or protein including an antibody is produced using various kinds of genetically-modified organisms including prokaryotic and eukaryotic cells. Many of the proteins used for medical treatment, research and the like are not suitable to be produced by prokaryotic cells such as bacteria because they are glycoproteins. For this reason, protein expression systems using eukaryotic cells such as yeast cells, insect cells or mammalian cells have been developed and widely used.
One of the major problems in the biotechnology for producing heterologous proteins is to produce and recover polypeptides, such as proteins and protein subunits), not easily expressed or secreted in genetically modified organisms. Since these proteins or protein subunits are expressed in cells at a very low level or a normal level, the scale of culturing and purifying tends to become larger in order to obtain a desired amount of proteins or protein subunits.
A typical method for solving such problems is to induce the proteins or protein subunits expressed in a cell to be secreted into a culture medium as high a level as possible. It is very useful even in purification to allow the proteins or protein subunits expressed in the cell to be secreted into an extracellular medium because these proteins are easily purified by doing so. In addition, the recombinant proteins or protein subunits secreted into an extracellular medium are advantageous in that protein decomposition occurring in the cell can be prevented and in that protein products with accurate folding can be obtained.
For successful secretion of the proteins expressed in a eukaryotic cell to the outside of the cell, a translocation of a protein traversing an intracellular endoplasmic reticulum is required. During the translocation, several modification steps required for protein activation occur concurrently, and thus the protein secreted to the outside of the cell can be considered as a mature protein which was immediately saccharified or modified.
Proteins secreted from a cell through a cell membrane are generally produced in the cell in the form of a precursor, and is referred to as a “preprotein”. The preprotein includes an additional peptide sequence at the amino terminal (NH3-terminal), and this peptide sequence allows the expressed protein to enter a secretion pathway by targeting this protein into an intracellular endoplasmic reticulum. This additional peptide sequence is referred to as a “protein secretion factor” or “signal sequence or signal peptide”.
In the case of a recombinant protein, secretion may not operate as expected because the natural signal sequence of the recombinant protein does not operate well in a host cell. Although there are many known signal sequences that can be used for the secretion of a specific recombinant protein, there is still a need for the discovery of additional signal sequences capable of promoting the effective secretion of recombinant proteins, particularly, immunoglobulins in a mammalian host cell.